Efficient spatially targeted gene editing using a near-infrared activatable protein-conjugated nanoparticle for brain applications

Spatial control of gene expression is critical to modulate cellular functions and deconstruct the function of individual genes in biological processes. Light-responsive gene-editing formulations have been recently developed; however, they have shown limited applicability in vivo due to poor tissue penetration, limited cellular transfection and the difficulty in evaluating the activity of the edited cells. Here, we report a formulation composed of upconversion nanoparticles conjugated with Cre recombinase enzyme through a photocleavable linker, and a lysosomotropic agent that facilitates endolysosomal escape. This formulation allows in vitro spatial control in gene editing after activation with near-infrared light. We further demonstrate the potential of this formulation in vivo through three different paradigms: (i) gene editing in neurogenic niches, (ii) gene editing in the ventral tegmental area to facilitate monitoring of edited cells by precise optogenetic control of reward and reinforcement, and (iii) gene editing in a localized brain region via a noninvasive administration route (i.e., intranasal).

(a-c) FTIR analyses of the UCNPs during the modification steps: citrate (cit)-coated UCNPs (black), silane modification with N3PTES and NH2PTES (red), conjugation of hydroxychloroquine (green) and conjugation of photo-cleavable linker (blue). Silanization at the surface of the UCNPs could be indicated by the emergence of the bands at 2937 cm -1 and 2894 cm -1 , corresponding to the asymmetric and symmetric stretching vibrations of CH 2 groups of the silane alkyl chain, while the bands at 1042 cm -1 and 1110 cm -1 are assigned to the vibration of Si-O-Si bonds. While the presence of N3PTES could be demonstrated by the strong band at 2105 cm -1 corresponding to the asymmetric stretch vibration of the azide group, the bands at 1452 cm -1 and 916 cm -1 indicating hydrogen bonding of NH 2 and N-H bending mode, respectively, ascribe to the presence of primary amines of NH2PTES. The attachment of HCQ to UCNPs was confirmed by the appearance of the band at 1513 cm -1 , ascribed to C-N stretching vibrations. Finally, the conjugation of PCL could be confirmed by the reduced peak attributed to the azide group (2105 cm -1 ), in contrast with the emergence of the bands around 2850 cm -1 (triazole) and 1560 cm -1 (N-O), indicating the successful cycloaddition of PCL with the azide groups. (d) Zeta potential analyses of UCNPs (0.5 mg/mL) in 1.0 mM KCl, pH 7. Results are average ± SEM, n=5. Silanization shifted the initially negative charge of the UCNPs (owing to citrate ions adsorbed to the NP surface) to a positive value due to the presence of amine groups from NH2PTES. Successful conjugation of HCQ decreased the number of available amine groups, shifting the NP surface charge back to a negative value. Further conjugation of PCL and Cre recombinase did not significantly affect the overall surface charge. Quantification of yttrium (Y) by ICP-MS analyses in fibroblasts exposed to different concentrations of Cre-UCNPs without HCQ. Cells were incubated with NPs (25 or 50 µg/mL) for a specific time, washed, harvested and finally freeze-dried. Concentration of yttrium was normalized per cell. Results are expressed as Mean ± SEM (n=3). (b) Cytotoxicity in fibroblasts exposed to a NIR laser for different times, as evaluated by ATP measurements at 48 h after laser exposure. Results are Mean ± SEM (n=3). Irradiation for up to 45 min did not significantly affect ATP production in irradiated cells.(c) Cytotoxicity of several concentrations of Cre-UCNPs (non-irradiated; with HCQ) in fibroblasts, as evaluated by ATP measurements. Fibroblasts were incubated for 4 h, washed, and ATP production was evaluated after 48 h. Results are Mean ± SEM (n=3-4 independent experiments). Cre-UCNPs were tolerated at concentrations ≤ 50 µg/mL, with minimal impact on cell metabolism. (d) Quantification of fibroblast number after incubation with Cre-UCNPs (25 or 50 µg/mL) for 4 h, washed, activated or not with a NIR laser, and cell number was evaluated at time 24 h. Results are expressed as Mean ± SEM (n = 3). No statistical significance was found after performing a two-way ANOVA test followed by Bonferroni's multiple comparisons test, suggesting that cell number was not affected after treatment with Cre-UCNPs at concentrations ≤ 50 μg/mL. (e) Cytotoxicity of UCNPs in fibroblasts evaluated by Annexin V/PI analyses. The percentage of apoptotic and necrotic cells was determined by flow cytometry. Fibroblasts were incubated with Cre-UCNPs (e and f) for 4 h, washed to remove the noninternalized UCNPs and finally allowed to grow for 24 h (e) or 48 h (f). Percentages of live (annexin V−/ PI−), early apoptotic (annexin V+/PI−), late apoptotic (annexin V+/PI+) and necrotic (annexin V−/PI+) cells were obtained. Results are expressed as Mean ± SEM (n = 3). Our results indicate that Cre-UCNPs at 25 µg/mL (even at 50 µg/mL) had no significant effect in cell apoptosis and necrosis. (g) Cytotoxicity of irradiated Cre-UCNPs in fibroblasts, as evaluated by ATP measurements. Fibroblasts were incubated with Cre-UCNPs (25 µg/mL) for 4 h, washed, irradiated for a specific time, and ATP production was evaluated after 48 h. Results are Mean ± SEM (n=3). No significant alterations were observed after irradiation for 15-30 min) with a NIR-laser (980 nm, 785 mW/cm -2 ). (a) Schematic representation of the experimental set up to monitor cell temperature after NIR exposure. Fibroblasts were incubated for 4 h with Cre-UCNPs (50 μg/mL), washed to remove the non-internalised NPs, and then exposed to a NIR laser (980 nm, 785 mW/cm 2 ; until 15 min of continuous irradiation). After irradiation, media was removed from the cells and they were analysed under a thermal camera. As a control, cells not exposed to Cre-UCNPs were irradiated for the same time (1, 2, 3, 4, 5, 10 and 15 min). (b) Temperature variation in cells alone or cells transfected with Cre-UCNPs after NIR laser irradiation (980 nm, 785 mW/cm 2 ) for different times. Results are expressed as Mean ± SEM (n = 3-5 wells per time and condition from 1 independent experiment). Our results indicate that the temperature of the cells increased approximately 5ºC during 15 min of irradiation, irrespective of the treatment with UCNPs. (c) Representative thermal camera images of cells incubated with Cre-UCNPs before and after NIR laser irradiation. (d and e) Colocalization of HSP70 with cell nuclei. Fibroblasts were incubated for 4 h with Cre-UCNPs (50 µg/mL), washed to remove the non-internalised NPs, and then exposed or not to a NIR laser (980 nm, 785 mW/cm 2 ; pulsed or continuous). Representative immunocytochemistry images of HSP70 expression in the different experimental groups, obtained from 3 independent experiments. Cells cultured in normal conditions present a diffuse pattern of HSP70 in their cytoplasm and in the nucleus. High colocalization between HSP70 and cell nuclei indicates a heat-stress response. Dashed line represents the average colocalization result for a heat shock of 43ºC of 1 h. Scale bar = 20 m. In (e), results are Mean ± SEM (n=3-6 wells per time and condition, 2 images per well). Statistical analyses were performed by one-way ANOVA followed by a Tukey´s multiple comparisons test. * denotes statistical significance (P=0.0484). No heat-shock stress was observed in cells after exposure to NIR laser (with or without Cre-UCNPs) for times ≤ 15 min. Our results suggest that heat-shock stress response can be prevented by applying a pulsed NIR activation (6 × 5 min, 5 min interval per pulse). For this purpose, fibroblasts were incubated for 4 h with Cre-UCNPs (50 µg/mL), washed to remove the non-internalised NPs, and then exposed or not to a NIR laser (980 nm, 785 mW/cm 2 ; pulsed or continuous until 30 min of irradiation). As positive control, cells irradiated with a 405 nm blue laser for 30 min. As negative control, cells not transfected with Cre-UCNPs were irradiated with a NIR laser for a specific time (i.e, 5, 10, 15 and 30 min). (a) Representative immunofluorescence images for γH2A.X on fibroblasts either transfected or not with Cre-UCNPs exposed to NIR laser for 30 min, obtained from a total of 12 images per condition. Scale bars = 25 m. (b) γH2A.X fluorescence intensity was normalized by the area of cell nuclei and expressed as a percentage of the positive control (i.e., cells irradiated with a 405 nm blue laser for 30 min). (c) Total number of γH2A.X foci (calculated by Image J, using the Find Maxima tool; noise tolerance: 50) normalized by the number of cell nuclei. In b and c, results are Mean ± SEM (n = 3 wells per condition, 4 microscopy images per well, each image had 425,000 m 2 ). Statistical analyses were performed by One-way ANOVA test followed by a Tukey´s multiple comparisons test. **** denotes statistical significance at P<0.0001. Our results indicate that UV light emitted by Cre-UCNPs cannot induce double-stranded DNA breaks on cells if they are exposed to NIR radiation (980 nm, 785 mW/cm 2 ) for less than 30 min. The DNA damage effect observed when irradiation time is above or equal to 30 min could be reduced in case of a pulsed NIR laser activation of Cre-UCNPs within cells (6x5 min). Results are presented as the average of 2 independent runs. (b) Transport of FITC-labeled transferrin known to selectively enter cells via clathrin-mediated endocytosis. Results are expressed as Mean ± SEM (n = 3). Statistical analysis was performed by one-way ANOVA followed by a Dunnett's multiple comparisons test, with *, ** denoting statistical significance (P=0.0117, P=0.0019, respectively). Dynasor and dansylcadaverine at concentration of 20 μM and 1 μM, respectively inhibited the internalization of transferrin (1 μg/mL) in fibroblasts. Cells were exposed to culture medium with or without dynasor/dansylcadavarine for 30 min, exposed to transferrin for 8 min, and finally characterized by flow cytometry. (c) Uptake of Cre-UCNPs (25 μg/mL) by fibroblasts in the presence of several endocytosis inhibitors. Cells were incubated with fluorescentlabeled Cre-UCNPs for 4 h in the presence of endocytosis chemical inhibitors, at non-cytotoxic concentrations, and then characterized the cells by flow cytometry. Results are Mean ± SEM (n = 3). Statistical analysis was performed by one-way ANOVA followed by a Dunnett's multiple comparisons test, with **, **** denoting statistical significance (P=0.0011, P<0.0001, respectively). Dynasor and dansylcadaverine treatment (clathrin-mediated endocytosis inhibitors) reduced the uptake of Cre-UCNPs by 46% and 66%, respectively, while cytochalasin-D (F-actin inhibitor) reduced 30% as compared to control cells. (d) qRT-PCR analyses showing the siRNA-mediated knockdown efficiency (24 h post-transfection with siRNA). Results are expressed as Mean ± SEM (n = 3). (e) Representative TEM images indicating the cellular uptake of Cre-UCNPs, obtained from a total of 18 images. NPs were found in vesicles of ~100 nm in diameter while others were being taken up by macropinocytosis. Black scale bar is 50 nm and white scale bar is 500 nm. Representative TEM images indicating the intracellular location of Cre-UCNPs (f) with and (g) without HCQ in fibroblasts, obtained from a total of 18 images. Cells were incubated with the NPs for 4 h. Scale bars = 1 µm (200 nm for insets i, ii, and iii). N: cell nucleus, C: cytoplasm, M: macropinocytosis, V: vacuole. Fibroblasts were incubated with Cre-UCNPs with or without immobilised HCQ (50 μg/mL) for different time points and then washed extensively, fixed, stained with a LAMP1 antibody, and characterised by confocal microscopy. For time 24 h in b.1 and b.2, cells were exposed for 4 h to Cre-UCNPs / Cre-HCQ-UCNPs, washed, cultured for additional 20 h and then processed has mentioned above. (a) Representative fluorescence images for LAMP1 staining in cells exposed for 4 h to Cre-UCNP with HCQ or Cre-UCNP without HCQ, obtained from a total of 18 images per condition. Scale bars = 50 µm (inset = 25 µm). (b) Number (b) and area (c) of LAMP1 foci per cell in cells exposed to Cre-UCNPs with or without HCQ. Results are expressed as Mean ± SEM (n = 3 wells, for each well 6 photos with an area of 41333 µm 2 were analyzed). **** denote statistical significance (P<0.0001) as assessed by one-way ANOVA followed by a Tukey´s multiple comparisons post-test. Number (d) and area (e) of Lysotracker positive foci per cell in cells exposed to Cre-UCNPs with or without HCQ. In this experiment, fibroblasts were stained with Lysotracker Red (50 nM) for 30 min. After the staining, cells were incubated with Cre-UCNPs with or without immobilized HCQ (50 µg/mL) for different time points and then washed extensively, fixed, and characterised by fluorescence microscopy. Results are Mean ± SEM (n = 3 wells, for each well 6 photos with an area of 41333 µm 2 were analyzed). No statistical significance was found after performing one-way ANOVA followed by a Tukey´s multiple comparisons post-test. Our results indicate that the incorporation of HCQ in Cre-UCNPs did not affect affect lysosomal pH, maintaining the number of Lysotracker (a pH-sensitive endolysosomal dye) puncta in fibroblasts over a period of up to 1 h after treatment.  C57BL/6J mice were euthanised followed by hair shaving and removal of dorsal skin and ribs, as well as the brain. Both tissues were fixed in 4% PFA and kept in cold PBS until further analyses. The thickness of the skin, ribs and whole brain was 0.5, 3 and 6 mm, respectively, as measured by a caliper. In the day of the experiment, the tissues were placed on top of the 96-well plate lid containing the Cre reporter fibroblasts transfected with Cre-UCNPs (50 µg/mL) for 4 h. The cells with the tissue barrier were then irradiated with a 980 nm laser at 785 mW/cm 2 for 3 cycles of 5 min. (b) Percentage of fibroblast cell recombination as measured by a high-content microscope though the evaluation of the intensity of GFP (at 48 h). Results are expressed as Mean ± SEM (n = 3 wells per each condition). (c) Percentage of power density after crossing skin (0.5 mm), ribs (3 mm) and the whole mice brain (6 mm). For the laser attenuation studies, each tissue was placed in a plastic petri dish on top of a thermal power sensor (Thorlabs, s310c). Both tissues were then irradiated with a 405 nm or a 980 nm lasers during 1 min. Laser attenuation values were calculated by normalising against laser power values obtained with the empty petri dish. The thickness of the tissues was measured by a caliper. Results are expressed as Mean ± SEM (n = 3 measurements per condition). in the muscle of C57BL/6 mice (approximately 4 mm deep from the skin). Immediately after transplantation, the site of injection was activated or not by a NIR laser at 980 nm (425 mW/cm 2 ; 3 cycles of 5 min irradiation) directly above the injection site and then allowed to recombine for 60 h. As a positive control, reporter cells were activated by a NIR laser in vitro and allowed to express the reporter for 48 h before transplantation. Cells without transfection with Cre-UCNPs were used as negative controls. (b) Representative immunofluorescence images of mouse muscle tissue transplanted with fibroblast reporter cells 60 h post-transplantation, obtained from a total of 36 images per condition. Scale bars = 30 µm. Total GFP intensity (c) and number of GFP-positive cells (d) in each section. Results are Mean ± SEM (n=5-6 animals, with 2 sections analyzed per animal and 3 images acquired in each section; the area of each photo was 45176 µm 2 ). **** denotes a statistical difference (P<0.0001) by one-way ANOVA test followed by a Bonferroni´s multiple comparisons test.  Recombination was observed to be dependent on NIR laser photoactivation. As a positive control neurons were infected with AAV9.ChR2-YFP for direct expression of ChR2 (i.e., independent of Cre recombinase). Scale bars = 50 µm.

Cell population Singlets YFP+
Cells vs Cre-UCNPs +NIR Supplementary Fig. 15 -Gating strategy for the definition of YFP + cells in Fig. 5b.

Supplementary Fig. 16 -Light-mediated currents measurements.
A cortical neuronal culture was used to validate blue light-dependent ChR2 depolarizations currents. After isolation cells were cultured for 7 days before treatments. AAV5.ChR2-YFP was added to the medium and let for ChR2-YFP expression for 7 days. (a) Voltage-clamp currents generated from blue-light stimulus. The current shows a peak current, characteristic of ChR2 depolarization. (b) Voltageclamp currents generated from NIR-light stimulus. A cortical neuronal culture without ChR2-YFP expression was used to validate NIR light-dependent depolarization currents. The current doesn't show the characteristic peak of ChR2 depolarizations. Animals were divided into two groups: the Cre-UCNPs+NIR group received the nanoparticles (30 µg) and NIR irradiation, while the sham group received only a saline solution injection in the same coordinates. Staining of IBA-1 (microglial marker) was performed 3 days after injections. (a) Representative images of IBA-1 staining, obtained from a total of 12 images per condition, showing the "injection path" ROI (ip) with high microglia activation and the VTA ROI defined for the analyses. Injection ROI was defined as a 200 µm square region surrounding "injection path" microglia activations. Scale bars = 1000 µm. (b) IBA-1 intensity was determined by fluorescence microscopy, and normalized to the ROI area. IBA-1 intensity was observed in the VTA targeted region. Results are expressed as Mean ± SEM (n=4 animals for Cre-UCNPs and 3 animals for Saline group, with 4 brain slices analyzed per animal). No statistical difference was observed after performing one-way ANOVA test followed by a Tukey´s multiple comparisons test. Quantification of Cre-UCNPs in the brain after intranasal administration. C57BL/6 mice were subjected to a single intranasal instillation of Cre-UCNPs and analysed by ICP-MS. Animals were divided into two groups: (i) Cre-UCNPs+NIR group (120 µg) and control group (saline). Yttrium content was determined by ICP-MS in the nasal cavity, olfactory bulb (OB), cortex and hippocampus, 4 h after administration. Results are expressed as % ID (initial dose of Y) per gram of tissue. Mean ± SEM (n=4 animals for Cre-UCNPs and 1 animal for control group). (c) Representative image of Cre-mediated recombination following NIR activation in only 1 side of the OB, obtained from 3 independent experiments. Recombination resulted in the expression of tdTomato, whose fluorescence was evaluated 4 weeks after administration. Scale bars = 50 µm. EPL: external plexiform layer, IPL: internal plexiform layer. (d) Quantification of tdTomato fluorescent areas in the NIR-exposed side was normalised to the unexposed side of the OB (noNIR), in order to validate the spatial resolution of NIR-mediated recombination. Results are expressed as Mean ± SEM (n= 3 animals treated with Cre-UCNPs and 2 with Saline; 6 images were obtained from each animal). Statistical analysis was performed by a two-tailed unpaired t test, with * denoting statistical significance (P = 0.0406).

SUPPLEMENTARY METHODS
Production of recombinant nlsCre. Recombinant nlsCre protein was purchased from X-PROT (Cantanhede, Portugal). Briefly, an in-house construct containing the nlsCre in frame with a GST tag (glutathione s-transferase) was cloned in a pGEX family vector for transfection in E. coli BL21codonplus strain (Agilent). Protein expression was induced with 0.5 mM isopropyl β-D-1thiogalactopyranoside and the process was carried out overnight at 28 ºC. After expression, bacterial cultures were centrifuged at 3000 g, for 15 min at 4 ºC and the sediment was resuspended in PBS supplement with 0.5% Triton X-100. Cells were mechanically lysed using an Emulsiflex device and the protein soluble fraction was recovered after a centrifugation step at 3000 g, for 20 min at 4 ºC.
The recombinant GST-nlsCre was then captured by affinity chromatography using glutathione stransferase 4B resin (GE healthcare) and the recombinant nlsCre was obtained after incubation with 10 U/mL thrombin (GE healthcare) for 16 h at 4 ºC with gentle mixing. The enzymatic digestion was stopped by adding to the mixture 0.5 M Na2HPO4 (1/5 volume) followed by an incubation for 3 h at room temperature. In order to improve protein purity, the eluate was diluted 5× in 20 mM HEPES buffer pH 7.7 and then loaded to a cationic exchange column Mono-S (GE healthcare). The purified nlsCre was eluted with a linear gradient of NaCl (0-1 M) in 20 mM HEPES pH 7.7 buffer at a 1 mL/min flow. Protein quantification was determined by absorbance reading at 280 nm using a NanoDrop apparatus and protein purity was estimated by SDS-PAGE with Coomassie staining.

NP-cell interactions: cytotoxicity.
For the cell viability assay, Cre reporter fibroblasts (1×10 4 cells/well) were seeded onto a 96-well plate and left to adhere. The cells were then incubated for 4 h with Cre-UCNPs (25, 50, 100, 250, 500 µg/mL), washed three times with cell medium to remove non-internalized NPs and cultured for additional 20 or 44 h. At these time points, ATP production was measured by a Celltiter-Glo Luminescent Cell Viability Assay (Promega) and cell number by a Neubauer chamber. For the annexin V/propidium iodide (PI) assay, fibroblasts (4×10 4 cells/well) were seeded on a 24 well plate and left to adhere overnight. The cells were then incubated with Cre-UCNPs (25, 50, 100, 250, 500 µg/mL) for 4 h, washed three times with warm PBS to remove noninternalized NPs, and incubated for additional 20 or 44 h. Then, the medium with the detached cells was collected and the adherent cells rinsed with PBS and trypsinized. Both the detached cells and adherent cells were then mixed and the cells were centrifuged for 3 min at 300 g. The cells were washed with PBS, resuspended in PBS (100 L), and stained with annexin binding buffer (Invitrogen, 200 µL, containing 2.5 L of annexin V-FITC conjugate). The cells were incubated in the dark at 8 room temperature for 15 min and then on ice before analysis in the flow cytometer. To stain the dead cells, PI (100 µL, 3 µg/mL) was added shortly before running each sample.
NP-cell interactions: heat-shock proteins. Cre reporter fibroblasts (4×10 4 cells/well) were incubated with Cre-UCNPs (50 µg/mL) for 4 h in serum-free DMEM medium and washed with warm PBS. Cells were irradiated for 5, 10, 15 or 30 min with NIR-light (785 mW/cm 2 , 980 nm). As a positive control, cells were incubated at 43ºC for 1 h. As negative controls, cells were incubated without Cre-UCNPs for 4 h and then irradiated (5, 10, 15 or 30 min) or incubated with Cre-UCNPs for 4 h and non-irradiated. Immediately after irradiation cells were fixed with 4% (v/v) paraformaldehyde and incubated with mouse anti-human HSP70 (3A3) antibody (1:50, sc-32239, Santa Cruz) for 1 h at room temperature. As a secondary antibody, cells were then incubated for 1 h in the dark with a secondary antibody sheep IgG-Cy3 anti-mouse (1:100, Sigma). Cell nuclei were stained with DAPI (Sigma) and the slides were mounted with mounting medium (Dako) and examined with a Zeiss LSM 710 confocal microscope using a Apochromat 40x/1.4 objective. Nuclear localization of HSP70 was quantified by colocalization between the nuclear staining (DAPI) and the HSP70 staining. This analysis was performed with the JACop plug-in for ImageJ through the Mander's overlap coefficient.
As negative control, cells were cultured in the absence of irradiation. At the end of all the procedures, the cells were fixed with 4% (v/v) paraformaldehyde, permeabilized with triton-X (0.3%, w/v) and blocked with 1% (w/v) BSA and 0.3 M glycine. Cells were then incubated with mouse anti γ-H2A.X (phosphor S139) antibody (ab11174, Abcam) (1:1000 in PBS containing 1% BSA) for 1 h at room temperature. As a secondary antibody, cells were incubated for 1 h in the dark with goat anti-rabbit IgG Alexa 488 antibody (1:100 dilution in PBS containing 1% BSA). Cell nuclei were stained with DAPI (Sigma), and the slides were mounted with mounting medium (Dako) and examined with a Zeiss LSM 710 confocal microscope using an oil immersion Apochromat 40x/1.4 objective.
NP cellular uptake and intracellular trafficking by TEM analyses. Cells were fixed by immersion in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer pH 7.4 solution for 5 days. After washing and 2 h in post-fixating 2% osmium tetroxide in 0.1 M sodium cacodylate buffer pH 7.4 solution, tissues were washed in buffer, incubated with 1% uranyl acetate overnight, washed in buffer and dehydrated through graded series of ethanol, and embedded in Epon (EMS).
Ultrathin sections were cut at 50 nm and prepared on a RMC Ultramicrotome (PowerTome, USA) using a diamond knife and recovered to 200 mesh Formvar Ni-grids, followed by 2% uranyl acetate and saturated lead citrate solution. Visualization was performed at 80 kV in a (JEOL JEM 1400 microscope (Japan)) and digital images were acquired using a CCD digital camera Orious 1100 W (Tokyo, Japan). The transmission electronic microscopy was performed at the HEMS core facility at i3S, University of Porto, Portugal.
In vitro Cre-mediated DNA recombination using a commercial transfection agent. Cre reporter fibroblasts (4×10 4 ) were seeded onto a 24-well plate and left to adhere overnight. Cells were incubated with different concentrations of Cre-UCNPs (1.5 -50 μg/mL) and Cre-UCNPs without HCQ (1.5 -50 μg/mL) for 4 h, washed with PBS, and left to grow in complete medium for 48 h. In a separate experiment, cells were incubated with nls-Cre recombinase (1.9 -750 nM) complexed with lipofectamine RNAiMAX (1.5 μL, Life Technologies) in antibiotic-free complete medium for 24 h.
After 24 h, the media was replaced with fresh full serum media. All complexing steps were performed at room temperature. Non-transfected cells were used as a negative control. For the assessment of positive cells percentage, flow cytometry was used. For this purpose, cells were washed with cold trypan blue solution, re-washed three times with cold PBS, dissociated with trypsin (0.1%, w/v, in PBS), centrifuged and ressuspended in PBS for flow cytometry analyses.
In vitro deep-tissue activation. Cre reporter fibroblasts (5×10 3 ) were seeded onto a 96-well plate and left to adhere overnight. The cells were incubated with Cre-UCNPs (50 g/mL) for 4 h, washed to remove the non-internalized NPs, and irradiated with a NIR laser (980 nm, 3 cycles of 5 min irradiation, at a power of 425 mW/cm 2 ). Photoactivation was performed through mice tissues of different thickness and composition: skin (0.5 mm), ribs (3 mm), whole brain (6 mm). Efficiency of laser penetration was evaluated overtime by direct analysis of GFP expression (mean green intensity in the cytoplasm of Cre reporter cells) in cells by the IN Cell Analyzer 2200 cell imaging system, using an inverted 20x PlanFluor objective/0.45 numerical aperture. Cell recombination was detected using an excitation filter 475/28 and an emission filter 511.5/23. Animal testing: isolation of SVZ cells. SVZ cell cultures were prepared from 1 to 3 days-old R26YFP mice. For this purpose, brains were removed from the skull and placed in Hank's balanced salt solution (HBSS) containing penicillin (100 U/mL) and streptomycin (100 μg/mL, Life Technologies). Coronal brain sections (450 μm thick) where obtained using a McIlwain tissue chopper and SVZ fragments were subsequently dissected. SVZ was digested in trypsin (0.025%, w/v, Life Technologies) and EDTA (0.265 mM, Life Technologies), followed by mechanical dissociation.